Products containing quantum of bioparticles and method for production thereof

ABSTRACT

A method for forming a substantially solid product containing a desired quantum of bioparticle, the method comprising (a) providing a sample of bioparticle in liquid form; (b) selecting a desired quantum of the bioparticle; and (c) forming a substantially solid product containing the desired quantum of the bioparticle, wherein the product is capable of being transferred between containers in its solid form without loss of bioparticle, and wherein the product is capable of releasing the bioparticle in a liquid.

TECHNICAL FIELD

The present invention relates to products containing a quantum ofbioparticles, particularly a quantum of microorganisms suitable for usein tests and assays.

BACKGROUND

There are many procedures performed in life sciences that involve themanipulation of small bioparticles such as cells, bacteria, viruses,protozoa, sperm, eggs, embryos and larvae. Generally the manipulation ofthese small bioparticles is inherently difficult because thebioparticles are too small to be visualised with the naked eye.

Where one is performing an experiment or procedure that involves addingbioparticles to a vessel (for example, a test tube) there is currentlyno simple technology available which allows one to know exactly, or atleast with a minimal degree of error, how many bioparticles have beenadded. Typically one would prepare a suspension of the bioparticles andthen perform an analysis (for example, enumeration by microscopy orculture on an agar plate), to estimate the number of bioparticles pervolume of liquid. An aliquot of this suspension, containing an estimatednumber of bioparticles, would then be used for a desired purpose; theexact number of bioparticles in the aliquot not being known.

In addition to problems associated with estimation of the numbers ofbioparticles by sampling, further problems may result duringmanipulation of the bioparticles in a particular procedure. For example,an unknown amount of the bioparticles are inevitably lost due to factorssuch as adhesion to surfaces of vessels or pipettes used, or todenaturation or death of some of the bioparticles. Further, bioparticlescan lose their viability or contents over time and accordingly productscontaining such materials may suffer from a short shelf life. Combinedwith the above problems, these factors may create gross inaccuracies inexperimental data.

A number of products are known which attempt to provide a standardisedproduct having a defined number of bioparticles. However, these productsunfortunately fail to address all of the problems above mentioned, andaccordingly, may be considered to fall short of providing a desirableproduct. For example, the degree of error in respect of the number ofbioparticles present from one sample of a product to another sample ofthe same product is likely to be in the order of greater than 50%, inmany cases the number of bioparticles present may vary 10 to 100 fold ormore.

One example of such known products is Cultiloops®) (Oxoid, Australia).Cultiloops® are disposable bacteriological culture loops that contain aloopful of freeze-dried culture of a specific microorganism and aregenerally used for quality control purposes in microbiologylaboratories. While Cultiloops® save time in the preparation of culturesfor quality control they unfortunately do not contain accurately definednumbers of cells per loopful. Further, it is possible that a number ofthe cells present may not be in a viable state.

Several companies supply vials containing an approximate number ofmicroorganisms in a freeze-dried form. Typically, these are manufacturedto an accuracy of 1 order of magnitude; for example, a vial will containbetween 1000 and 10000 bacteria. To use these products, one generallyadds water to the vial to resuspend the freeze-dried microorganisms,subsequently using a pipette to transfer the microorganisms to a sample.Due to the nature of this product, and the means by which it is used, itmay not be considered to adequately address the issue of providingaccurate and consistent numbers of bacteria, or the issue of the loss ofunknown quantities of bacteria during manipulation as a result ofadhesion to the side of the vial or the pipette.

BTF Pty Ltd (Australia) markets a product known as EasySeed™ C&G thatprovides an accurately defined number of inactivated Cryptosporidium andGiardia in liquid in a test tube. While it may be considered that thisproduct overcomes many of the issues associated with providing accuratenumbers of microorganisms, during use of the product, an unknown numberof the Cryptosporidium and Giardia are generally lost due to adhesion tothe side of the test tube or pipette. Further, the cells are notprovided in a viable state.

A further example of a presently available product are lenticles,freeze-dried quality control samples prepared by the UK Public HealthLaboratory Service (PHLS). Lenticles are prepared by pipetting drops ofa viscous bacterial culture a surface and drying the drops to form alens-shaped freeze-dried pellet. While the lenticules may be consideredto overcome the inaccuracies associated with handling liquid qualitycontrol samples, they unfortunately do not contain accurately definednumbers of bacteria.

A further product, known as TrueCount® (Becton Dickinson, San Jose,USA), is used in conjunction with flow cytometry to allow one todetermine the number of specific cells per millilitre of blood, forexample. The product consists of dried balls of approximately 1 mmdiameter that contain approximately 50,000 fluorescent beads ofapproximately 5 μm diameter. While this product may overcome problemsassociated with the loss of materials during manipulation of a liquidsample, it does not contain an accurately defined number of beads withinthe dried ball. Further, as the beads do not representbiological-derived material, the product, and the procedure of producingthe product, is not concerned with, and therefor may not adequatelyaddress, the issue of accuracy or maintenance of viability of thematerials.

U.S. Pat. No. 3,932,943 describes a process for the production of ahomogeneous, lyophilised product containing at least one biologicallyactive component. The process involves spraying a solution or colloidalsuspension containing the biologically active component into a movingbath of fluorocarbon refrigerant, and subsequently lyophilising theresultant frozen droplets. The inventors report that the product has aspherical shape, free-flowing properties, and rapid dissolution times.However, the process does not address the issue of preparing a productthat contains accurately defined numbers of bioparticles. In addition,it may be considered that this process does not adequately address theissue of maintenance of bioparticle viability, especially where suchbioparticle is a cell.

U.S. Pat. No. 6,106,836 describes a process for the production of avaccine product comprising a container with freeze-dried vaccinecomponents therein. The process involves the formation of ballscontaining biological components of estimated numbers utilising thesteps of freezing droplets of a suspension containing the biologicalcomponents in a cryogenic liquid and subjecting them to freeze-drying.The process of this patent does not immediately address the issue ofpreparing a freeze-dried product that contains accurate numbers ofbioparticles. By contrast, products containing estimated numbers ofcomponents are made via the above mentioned process, their titremeasured, and then a number of products combined, or used to supplementanother product, to obtain a desired quantity of components. Inaddition, the process of U.S. Pat. No. 6,106,836 may not be consideredto adequately address the issue of maintenance of the viability ofbioparticles during processing. Rather, the process centres on the lossof viability of the bioparticles followed by supplementation of theresultant product with additional viable materials.

Further, U.S. Pat. No. 3,655,838 describes a method for the preparationof pelletised reagents purportedly in a stable, accurate form. In thismethod, a suspension containing predetermined concentrations of desiredreagents is formed into calibrated droplets which are allowed to fallinto a liquid having certain characteristics, one of which is atemperature gradient suitable to freeze the droplets. Subsequently, thedroplets are dried to form the pelletised product. While the method aimsto provide products containing predetermined and pre-tested measuredamounts of certain reagents, it may be considered to suffer frominaccuracies in the actual concentration or number of specificcomponents present, due to methods employed to arrive at initialconcentration values. Further, the method may be considered not toaccurately address the issue of maintenance of viability where thereagent to be processed is a bioparticle.

Current methods for preparing DNA and protein standards for example,typically rely on measuring the absorbance of a solution of DNA orprotein and calculating the concentration and then diluting the solutionto the desired concentration. These methods do not provide accuratestandards.

The present inventors have now developed methods which are capable ofproducing a desired quanta of bioparticles in products suitable for useas accurate standards for a variety of biological and analyticalapplications.

SUMMARY OF INVENTION

The present inventors have devised processes which surprisingly allowfor the preparation of a substantially solid product containing adesired small quantum or number of microorganisms in a stable formatthat can be easily manipulated while minimising the loss of any of themicroorganisms. The process is particularly applicable to the formationof a product comprising an accurate small number of viable or stablemicroorganisms. It is believed that the nature of the product accordingto the invention will allow for simplified manipulation ofmicroorganisms and for more accurate and reproducible results fromprocedures utilising the microorganisms.

In a first aspect, the present invention provides a process for forminga product containing a desired number of microorganisms, the methodcomprising: (a) providing microorganisms; (b) selecting a desired numberof the microorganisms using means to sense a bioparticle; and (c)forming a substantially solid product containing the desired number ofmicroorganisms, wherein the product is capable of being transferredbetween containers in its solid form without loss of any microorganism,and wherein the product is capable of releasing the microorganism in aliquid.

In a second aspect, the present invention provides a process for forminga product containing a defined number of microorganisms, the processcomprising: (a) providing microorganisms selected from the groupconsisting of microorganisms, cells, vectors, particles containing abiological material, and mixtures thereof in suspension; (b) selecting adefined number of the microorganisms of between 1 and 1000 from thesuspension by means to sense a microorganism; (c) capturing the definednumber of microorganisms in a frozen body; and (d) drying the frozenbody to produce a product containing the defined number ofmicroorganisms, wherein the product is capable of being transferredbetween containers in its solid form without substantial loss of themicroorganisms, wherein the product is capable of releasing themicroorganisms in a liquid, and wherein the defined number of themicroorganisms in the product when measured in two or more replicates iswithin a standard deviation of less than about 10% of the definednumber.

In a third aspect, the present invention provides a substantially solidproduct containing a desired number of microorganisms of between 1 and1000, wherein the product is capable of being transferred betweencontainers in its solid form without loss of any microorganism, andwherein, the product is capable of releasing the quantum ofmicroorganism in a liquid.

In a fourth aspect, the present invention provides a product containinga quantum of microorganism produced by the process according to thefirst or second aspects of the present invention.

In a fifth aspect, the present invention provides use of a productcontaining a quantum of microorganism according to the third or fourthaspects of the present invention in an assay or test.

In a sixth aspect, the present invention provides use of a productcontaining a quantum of microorganism according to the fifth aspect ofthe present invention in an assay or test.

In a preferred form, the microorganism is selected from bacteria, cells,vectors, particles containing a biological material, and mixturesthereof. The microorganisms can be viruses, bacteria, yeast, fungi,parasites or protozoa, the cells can be plant cells, animal cells, orgametes, the vectors can be plasmids or viroids, and the particles canbe beads, for example that contain protein, peptides, polysaccharides,nucleic acid or mixtures thereof bound to the surface of the particlefor example.

Preferably, the microorganisms are selected from the group consisting ofLegionella, Salmonella, Leptospirosis, Escherichia, Saccharomyces,Clostridium, Vibrio, Pseudomonas, Bacillus, Streptomyces,Staphylococcus, Enterobacter, Listeria, Candida, Zygosaccharomyces, andmixtures thereof. More preferably, the bacteria are Escherichia coli orBacillus cereus.

Preferably, the parasites are selected from Cryptosporidium, Giardia,Cyclospora, Toxoplasma, Eimeria and mixtures thereof.

The bioparticle may also be in the form of particle containing or havingbiologically derived material such as protein, peptide, carbohydrate,polysaccharide, peptide, nucleic acid or mixtures thereof.

Further examples of bioparticles are blood cells, human imunodeficiencyvirus (HIV), Norwalk virus, herpes simplex virus.

A product according to the invention may comprise a single species ofbioparticle, or alternatively, two or more species of bioparticle.

When the bioparticle is a living organism, the product preferablycontains a quantum of viable organisms. In this form, the product isparticularly suitable for use as a quality control in microbialcultures. It will be appreciated, however, that there are othersituations where viability is not necessary for the product. Forexample, the product may include a quantum of proteins or nucleic acidswhich are useful for biochemical or molecular biology standards. Theprotein may be an enzyme which is required at a desired amount for anassay. Similarly, the nucleic acid may be a quantum of a gene or nucleicacid probe which can be used as a molecular biological tool in an assay.

In a preferred form, the sample of bioparticle is in liquid form.Examples include but not limited to microbial cultures, suspensions ofcells, suspensions of particles including one or more bioparticles. Thepresent inventors have found that a flow cytometer is particularlysuitable for selecting and capturing a quantum of the bioparticle inliquid form. In addition, the selecting and capturing a quantum of thebioparticle in liquid form can be carried out using any accuratemeasuring device. Examples include pipettes, micro-pipettes and othermicro-metering devices. Importantly, the device or step should notretain or adversely affect the capture of the desired quantum ofbioparticle.

In a preferred form, the quantum of bioparticle is selected byaccurately counting a desired number of bioparticle units and capturingthe desired number in a defined volume. The defined volume is usuallyless than about 1 ml, usually less than 0.1 ml, and typically about 0.03ml.

Preferably, a solid body is formed by freezing, preferably snap-freezinga volume of liquid containing the quantum of bioparticle and then dryingthe frozen body to form the product. One preferred form is carried outby placing the defined volume, usually a droplet, into a cryogenicliquid. The cryogenic liquid can be liquid nitrogen, liquid helium orliquid oxygen. More preferably, the cryogenic liquid is liquid nitrogen.The frozen liquid is then preferably processed by freeze-drying to forma substantially solid product, usually as a small roundish mass in theform of a ball or a pellet.

In another preferred form, the product is produced without freezing. Forexample, a drop or aliquot containing the bioparticle could be droppedor placed onto a surface and then dried. In one form, the material couldbe placed on an absorbent material and then dried.

In one preferred form, the cryogenic liquid is placed in a container anda droplet is placed in the container to form a frozen body. Thecontainer holding the frozen body is then subjected to freeze drying toform a substantially dry solid product within the container. Afterdrying, the container can be capped or sealed for storage and transportof the product. To provide an environment that will maintain thestability of bioparticle if required, the container can be capped orsealed whilst under vacuum or it can be filled with an inert gas. Inthis form, the quantum of bioparticle is captured within the dry solidproduct which can be free to move within the container. The product canbe manipulated in its dry state without loss of any bioparticle to thecontainer or its surroundings.

Another advantage of the present invention is that the desired quantumof bioparticle can be accurately dispensed. When measured in two or morereplicates, the number of the bioparticle in the product is within astandard deviation of less than about 20%, preferably less than about15%, more preferably less than 10%, and even more preferably less thanor around 5%. The method according to the present invention is capableof consistently providing a number of products having a quantum ofbioparticle with an accuracy of between about 4% and 8%.

The present invention is suitable for producing a large number ofproducts having the same desired quantum of bioparticles. The method issuitable for automation to produce large numbers of products in a batch,for example.

The method according to the present invention is suitable for providingsmall numbers of bioparticle of less than about 10⁵. The methodaccording to the present invention is particularly suitable forproviding small numbers of bioparticle of less than about 10⁴,preferably less than about 10³. In particular, the method can accuratelyprovide numbers of bioparticle of less than about 100. This achievementis quite remarkable when considering that the bioparticle can be cellsor microorganisms in relatively large volumes compared to the actualsize of the bioparticle.

In one preferred form, the method further includes the step of selectinga desired bioparticle type from a mixture of different bioparticles. Theselection may be on any suitable physical characteristic of abioparticle in solution. Examples include, but not limited to size,shape, colour, fluorescence, viability, electrorotation, impedence,refractivity, light scattering, time of flight through a laser beam,mass or charge. Characteristics can also be measured by reacting thebioparticles with a molecule that responds to certain properties withinthe bioparticle. For example, a fluorogenic substrate such as fluoreseindiacetate that becomes fluorescent in the presence of esterase activitywithin the cell. Other examples include, but not limited to nucleic acidstains such as propidium iodide and syto 16, the cell respiration stain5-cyano-2,3-ditolyl tetrazolium chloride (CTC), enzymatic substrates,lectins, antibodies, DNA probes.

Preferably, one freeze-dried product is formed per quantum of desiredbioparticles to form the product. Alternatively, two or morefreeze-dried products are formed per quantum of desired bioparticles toform the product.

Preferably, the method further comprises a final quality control step.Preferably, the quality control step involves one or more of:

-   -   counting the number of bioparticles contained within one or more        products; and    -   measurement of the uniformity of the product, by weighing or        measuring the size of one or more products.

Preferably, the quantum of bioparticles comprises a single species ortype of bioparticle. Alternatively, the quantum of bioparticlescomprises a mixture of two or more species or types of bioparticle.

Each product may additionally comprise supplementary agents. In oneform, the supplementary agents are those which aid in maintainingviability or stability of the bioparticle. More preferably, thesupplementary agents are cryopreservatives.

Other suitable supplementary agents include, but not limited to one ormore of sucrose, trehalose, lactose, maltose, glucose, galactose,raffinose, fructose, xylose, cellobiose, gelatin, xantham gum, guar gum,maltodextrans, polyethylene glycol, dextran, polyvinyl pyrrolidone,sodium thiosulfate, activated charcoal, ascorbic acid, ascorbateperoxidase, glutathione reductase, peroxiredoxin, sodium glutamate,proline, potassium glutamate, proline betaine, glycine betaine, skimmilk, serum, trypsin, peptone, tryptone, yeast extract, soy protein,meat extract, mannitol, glycerol, sorbitol, inositol, butanol, tertiarybutyl alcohol, honey, sodium acetate, myo-inositol, calcium chloride,whey, hydroxyectoine, and ectoine.

In one preferred embodiment, the present invention provides a method forforming a product containing a desired quantum of viable bacteria, themethod comprising:

-   (a) providing a sample of bacteria in liquid form;-   (b) selecting a quantum of the bacteria in a defined volume using a    cytometer;-   (c) adding the defined volume of the bacteria as a drop to a    cryogenic fluid to form a frozen ball containing the quantum of the    bacteria; and-   (d) subjecting the frozen ball to freeze-drying to form a    substantially dry solid product containing the quantum of the    bacteria, wherein the product is capable of being transferred    between containers in its solid form without loss of the bacteria,    and wherein the product is capable of releasing the quantum of    viable bacteria in a liquid.

Preferably, the bacteria are selected from the group consisting ofLegionella, Salmonella, Leptospirosis, Escherichia, Saccharomyces,Clostridium, Vibrio, Pseudomonas, Bacillus, Streptomyces,Staphylococcus, Enterobacter, Listeria, and mixtures thereof. Morepreferably, the bacteria are Escherichia coli or Bacillus cereus. Themicroorganism may also be a yeast such as Candida and Zygosaccharomyces.

In another preferred embodiment, the present invention provides a methodfor forming a product containing a quantum of a protein, carbohydrate,polysaccharide, gene or a nucleic acid molecule, the method comprising:

-   (a) providing a sample of a bioparticle containing a known number of    copies of a protein, carbohydrate, polysaccharide, gene or nucleic    acid molecule in liquid form;-   (b) selecting a quantum of the bioparticle in a defined volume using    a cytometer;-   (c) adding the defined volume of the bioparticle as a drop to a    cryogenic fluid to form a frozen ball containing the quantum of the    bioparticle; and-   (d) subjecting the frozen ball to freeze-drying to form a    substantially dry solid product containing the quantum of the    bioparticle, wherein the product is capable of being transferred    between containers in its solid form without substantial loss of the    bioparticle, and wherein the product is capable of releasing the    quantum of the bioparticle containing the quantum of the a protein,    carbohydrate, polysaccharide, gene or a nucleic acid molecule in a    liquid.

Preferably, the bioparticle is selected from microorganism, cell orparticle containing the protein, carbohydrate, polysaccharide, gene ornucleic acid molecule.

For example, a gene of interest together with a reporter gene having adetectable gene product can be added to a bacterium under the control ofthe same promoter. Only bacteria that produce the detectable reportergene product can be selected which would express a defined level of bothgenes. Thus a defined amount of the gene of interest would be producedin the product.

Preferably the bioparticle is microorganism containing a gene or nucleicacid molecule.

Throughout this specification, unless the context requires otherwise,the word “comprise”, or variations such as “comprises” or “comprising”,will be understood to imply the inclusion of a stated element, integeror step, or group of elements, integers or steps, but not the exclusionof any other element, integer or step, or group of elements, integers orsteps.

Any discussion of documents, acts, materials, devices, articles or thelike which has been included in the present specification is solely forthe purpose of providing a context for the present invention. It is notto be taken as an admission that any or all of these matters form partof the prior art base or were common general knowledge in the fieldrelevant to the present invention as it existed in Australia before thepriority date of each claim of this application.

In order that the present invention may be more clearly understood,preferred forms will be described with reference to the followingdrawings and examples.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a schematic diagram of a preferred embodiment of thepresent invention in the form of a ball.

MODE(S) FOR CARRYING OUT THE INVENTION

The invention relates to a process for preparing a product whichcontains a desired quantum of bioparticle. The product of the inventiongenerally comes in the form of a single mass, preferably in the form ofa ball or pellet. However, the inventors contemplate the product alsocomprising a number of balls or pellets, preferably 3 to 10 balls orpellets, which taken together provide the quantum of the bioparticle.While the following description focuses on the embodiment of theinvention in which the product represents a single ball, it will beappreciated by skilled persons in the art that the description may bereadily read to encompass the embodiment in which the product comprisesa group of balls, or any other forms of a substantially solid mass.

In order to facilitate manipulation of the product following formationthereof, the ball is preferably large enough to be visible to the humaneye. Accordingly, the product of the invention is preferably about 0.1to 10 mm in diameter or width, and more preferably about 4 mm. However,it will be appreciated that the product may have a diameter orcross-sectional width larger than 10 mm, or of a diameter orcross-sectional width smaller than 0.1 mm.

As used herein, the term “bioparticles” refers to particles of abiological nature. Such bioparticles may preferably be cells, ofprokaryotic or eukaryotic origin, or alternatively, proteins or cellorganelles such as nuclei, viruses, viroids, plasmids, or vectorscontaining known nucleic acid material. Accordingly, bioparticles whichmay be applicable to the invention include, bacteria, fungi, parasites,yeast or virus, single or multi-cellular protozoa, or prions. Examplesof such bioparticles include Legionella, Salmonella, Leptospirosis,Escherichia, Saccharomyces, Clostridium, Vibrio, Pseudomonas, Bacillus,Streptomyces, Staphylococcus, Enterobacter, Listeria, Candida,Zygosaccharomyces, Cryptosporidium, Giardia, Cyclospora, Toxoplasma,Eimeria, blood cells, HIV, Norwalk virus, herpes simplex virus. Thebioparticle may also be in the form of particle containing or havingbiologically derived material such as protein, peptide, carbohydrate,polysaccharide, peptide, nucleic acid or mixtures thereof. A productaccording to the invention may comprise a single species of bioparticle,or alternatively, two or more species of bioparticle.

As previously mentioned, the invention is particularly applicable to theformation of a product comprising viable bioparticles. As used herein“viable bioparticles” are materials capable of working, functioning, ordeveloping substantially as in their native state, or a state to whichthey have been designed to have via laboratory manipulation.Accordingly, in the case of cells, “viable bioparticles” are thosematerials capable of, for example, functioning, growing, developing and,where applicable, infecting a host.

As used herein a “quantum” of bioparticles refers to the number ofbioparticles present, or desired to be present, within the product ofthe invention. A “quantum” does not necessarily imply that exact numbersof bioparticles will be present within a sample or product according tothe invention without some small degree of variation. Typically, whenmeasured in two or more replicates, the number of the bioparticles inthe product is within a standard deviation of less than about 20%, morepreferably less than about 10%. In practice, the present invention candeliver a quantum of bioparticle in a product with an accuracy notpreviously achieved consistently by other methods. For example, smallnumbers of less than about 100 bioparticles can readily be provided withan accuracy of less than about 7% standard deviation. Generally,“quantum” of bioparticles, refers to the number of bioparticlesestimated to be present via conventional means used in the art; forexample, enumeration by microscopy or culture.

According to the invention, a “quantum” of bioparticles, is the numberof bioparticles present in the product of the invention. Preferably, a“quantum” represents an exact number of bioparticles. However, theinventors contemplate a minimal degree of error in the number ofbioparticles. The inventors believe such error may be monitored andminimised by conducting quality control checks of the products formed inthe method of the invention.

It will be appreciated that the number of bioparticles selected to bepresent within the product of the invention may vary depending on thenature of the bioparticles to be processed, the desired size of thefinal product (the freeze-dried balls for example), and the ultimate useto which the final product will be put. For example, a productcontaining Escherichia coli, which is desired to be used in qualitycontrol of microbiology culture media, will preferably contain a smallnumber, typically 30 viable E. coli cells. In addition, a productaccording to the invention may comprise a single species of bioparticle,or alternatively, a mixture of two or more specific species or types ofbioparticles.

The product of the present invention preferably contains constituents inaddition to the quantum of bioparticles. For microorganisms, the“constituents” will generally comprise those components of culture mediain which the microorganisms were grown or suspended in prior toprocessing according to the invention. Such constituents will be readilyrecognised by persons of general skill in the field, and will becomefurther apparent from the description of the method of the invention tofollow. The product of the invention may additionally containsupplementary agents which may have been introduced during one or moresteps of the process, or added to the growth or suspension media inwhich the microorganisms were initially prepared.

“Supplementary agents” are those agents which may aid in thepreservation of the viability or stability of the bioparticles duringprocessing (for example, cryopreservatives such as glycerol or dimethylsulfoxide (DMSO), antioxidants such as activated charcoal, sugars suchas glucose). Alternatively, agents which may be desired to be present inthe final product based on a particular application to which it will beput. For example, a dye may be added so that when, in use, the productis added to a liquid sample, a colour change of the sample will occur.Similarly, a detergent may be added to assist with re-hydration, orcommon bulking agents may be used to help give body to the resultantproducts of the invention. Such agents may be introduced at variousstages during the method of the invention. It is preferable, howeverthat at least some agents be added at the initial stage of preparing thebioparticles.

The inventors particularly contemplate the presence of cryopreservativeagents within the product of the invention. Such appropriatecryopreservative agents, which are well known in the art, includeglycerol, sugars which may be used at a concentration of between about 1and 20% (w/v), or dimethyl sulfoxide (DMSO), which may be used at aconcentration of between about 1 and 20% (v/v).

The inventors also contemplate the presence, within the products of theinvention, of protective agents that assist the survival or maintenanceof the viability or stability of the bioparticles during the dryingprocess. Such appropriate protective agents include, but not limited to,activated charcoal, honey, sodium glutamate, raffinose and animal serum.Skilled persons may be able to identify further appropriate protectiveagents. These protective agents may be used at concentrations of betweenabout 1 and 30% (v/v), for example.

It will be appreciated that the cryopreservative and the protectiveagents used in the invention may be varied so as to obtain the mostoptimal conditions for the particular bioparticle to be processed. Byway of example, where the bioparticles represent bacteria such as E.coli, appropriate cryopreservatives include glycerol and DMSO andappropriate protective agents include activated charcoal.

An example of a product according to the present invention in the formof a ball and its use is shown in FIGS. 1A to 1C. In this embodiment,the solid product is in the form of a substantially solid freeze-driedball 10 provided in a vial 12 (FIG. 1A). The vial 12 is sealed with astopper 14 for convenient storage and transport. It will be appreciatedthat other forms of storage would also be suitable for the solidproduct. The ball 10 in its solid form is transferable from the vial 12to another container without the concern of any loss of the bioparticlefrom the ball 10. As the bioparticle is contained in or associated withthe ball, portions of the bioparticle cannot be left in a container whentransferred to some other container.

FIG. 1B shows an example of dispensing the ball 10 from the vial 12 to asolid microbial culture medium 16 in a petri dish 18 for subsequentculture of the bioparticle in the form of a microorganism. The stopper14 is removed from the vial 12 and the ball 10 is tipped onto thesurface of the culture medium 16. When the ball 10 is removed from thevial 12, there will be no microorganism or other bioparticle present orleft over in the vial 12. The microorganisms in the ball 10 can then beobtained by adding any suitable liquid. In FIG. 1B, liquid in the formof drops of sterile water 20 are added to the ball 10 on the medium 16using a pipette 22. The ball 10 then dissolves releasing themicroorganisms onto the medium 16. The petri dish 18 can then beincubated at a desired temperature for a period of time for themicroorganisms to grow. Each microorganism will grow forming discretecolonies 24 on the medium 16 as indicated in FIG. 1C.

Methods

A preferred embodiment of the invention is described in terms of theformation of a product in the form of a single ball comprising a quantumof one species of bioparticle in the form of a microorganism. It shouldbe appreciated however, that the embodiment is readily applicable to aproduct comprising a group of balls or other solid forms, which takentogether provide the quantum of the same or different type ofbioparticle.

In general terms, a preferred method for forming freeze-dried balls ofthe invention comprises the following steps

-   (a) providing a sample of bioparticle in liquid form;-   (b) selecting a quantum of the bioparticle in a defined volume;-   (c) freezing the defined volume of liquid to form a frozen ball    containing the quantum of the bioparticle;-   (d) drying the frozen ball to form a substantially dry solid product    in the form of a freeze-dried ball containing the quantum of the    bioparticle, wherein the ball is capable of being transferred    between containers in its solid form without loss of any    bioparticle, and wherein the ball is capable of releasing the    bioparticle in a liquid; and optionally-   (e) carrying out one or more quality control steps on the    freeze-dried product.

Each step of the preferred process will be described more fully below.

Providing a Sample of Bioparticle in Liquid Form

The first step of the method of the invention involves preparing andproviding a sample of bioparticle in a liquid suspension media.

Generally, the bioparticle will be prepared according to methodologywell known in the technical field to which the invention relates. Forexample, in the case of prokaryotic or eukaryotic cells, the cells maybe grown in an appropriate liquid growth media (for example, nutrientbroth (Oxoid, Australia) in the case of E. coli) to a desired density.In any case, it will be appreciated that standard texts referred to inthe art will provide information of appropriate means for providingvarious bioparticles of use in the invention.

As will be appreciated, the liquid growth media referred to above willvary depending on the nature of the bioparticle to be processed; and theultimate use to which the product of the invention will be put. Forexample, where the bioparticle represents cells, the media in which theyhave been grown may represent the suspension media. Alternatively, inthe case of cells, they may be harvested from growth media andresuspended in an alternative media, which may be more suitable, havingregard to the use to which the final product will be put. In any case,those persons of skill in the art may readily be able to identifysuitable suspension media for a particular bioparticle based onwell-known principles in the art and by reference to standard texts.

The preparation of a bioparticle in the form of a microorganism forsubsequent culturing or growth should preferably aim to maximisemaintenance of viability or stability of the microorganism throughoutthe subsequent manufacturing process. Skilled persons will be able torecognise such conditions. However, by way of general example, theoptimisation of conditions at this stage of the manufacturing processmay involve growth of the microorganism under specific conditions thatenhance robustness. For example, growth of the microorganism to aparticular stage of growth known, or found, to be optimal for survivalof a particular microorganism. Alternatively, exposure of themicroorganism to conditions such as starvation or heat shock may beadvisable. By way of specific example, E. coli cells that are in thestationary growth phase will maintain their viability better than cellsin logarithmic growth phase. Bacteria such as Legionella may bettermaintain viability through the manufacturing process if they have beenstarved for several days by storage in distilled water. There are manyother situations or conditions for other microorganisms well known tothe art that would also be applicable for the present invention.

The inventors contemplate that maintenance of the viability of themicroorganisms during subsequent processing in the invention, may beenhanced by introducing supplementary agents to the growth and/orsuspension media within which the microorganisms may be grown and/orsuspended. The addition of supplementary agents to the growth and/orsuspension media for alternative reasons is also contemplated by theinventors. Suitable supplementary agents are described above.

In addition, the inventors contemplate that the microorganisms may bemodified during the initial preparation step. Such modification may, forexample, enhance subsequent analysis and sorting or alternatively, mayprovide desirable characteristics suited to the ultimate use to whichthe product of the invention will be put. For example, cells may be inrecombinant form, or may be labelled or tagged with suitable agents (forexample, antibodies, fluorescent stains such as fluorescein diacetate(FDA), propidium iodide (PI), oligonucleotides).

Selecting a Quantum of the Bioparticle in a Defined Volume

The second step of the manufacturing process of this preferredembodiment of the invention involves manipulating the liquid samplecontaining the bioparticles using flow cytometry techniques known in theart (Shapiro, H. M. (1990) Flow Cytometry in laboratory microbiology:new directions. American Society for Microbiology News 56, 584-588;Shapiro, H. M. (1995) A practical guide to flow cytometry, thirdedition. A. R. Liss, New York). By using such technology, desiredbioparticles may be identified and selected. Such “desired bioparticles”are materials of a desired nature or character. For example, aparticular cell type which may be present in a background of alternativebioparticles, or those cells of a certain type which are viable.

As will be appreciated, the technique of flow cytometry may be used toanalyse and select bioparticles based on preselected physicalcharacteristics. For example, absorption at a particular wavelength,density, magnetism, specific gravity, impedance, ability to scatterlight, luminescence, or fluorescence. In addition, the inventorscontemplate the use of Coulter sensing and raman microscopy inconjunction with flow cytometry to analyse and sort bioparticlesaccording to the invention.

While a single specific preselected characteristic of a particularbioparticle may be used for analysis or selection thereof in this stepof the manufacturing procedure of the invention, the inventors alsocontemplate the use of any other suitable method or device for selectinga number of bioparticles from a liquid sample. Examples include pipette,micro-pipette, metering devices and multiparameter flow cytometry.“Multiparameter flow cytometry” is used herein to mean the measurementof several characteristics of a particular bioparticle to differentiateit from another.

The techniques available to select bioparticles using flow cytometry arewell documented in the art. However, by way of exemplification, whereone wishes to rely on optical analysis, a sample containing bioparticlesmay be mixed with one or more dyes, fluorescent or luminescent agents,or antibodies conjugated to such agents prior to analysis via flowcytometry so that specific bioparticles are stained or tagged. Anexample of such a process involves the use of the dye fluoresceindiacetate which specifically stains viable cells. Following stainingwith fluorescein diacetate, the sample may be analysed and single viablebioparticles that are at a specific stage of their life cycleidentified.

The desired bioparticles may be selected, or sorted, from the sample, onthe basis of the preselected characteristics mentioned herein before,and in accordance with flow cytometry technology. Utilising the flowcytometer, a quantum of a desired material, or materials, may be sortedor captured.

In accordance with this step of the manufacturing process, the flowcytometry apparatus may express a volume of liquid containing thequantum. Preferably, the apparatus is configured such that the liquidcontaining a quantum of the bioparticles is forced through an orifice ina downward direction so that a droplet is formed. The size of theorifice can be changed to precisely control the volume of the droplet.

While the above technique is considered by the inventors to be apreferred means for material sorting and formation of a volume of liquidcomprising a quantum of selected bioparticles, it is contemplated thatalternative means known in the art may be used. For example, theinvention may employ piezo capillary dispensing, a piezo-actuatedcatcher tube, charged droplet deflection, acoustic manipulation(Standing Wave, Shock Wave), electrostatic manipulation, opticaltweezers, pipettes, micro-pipettes or other metering devices. Those ofgeneral skill in the art may realise further techniques may be readilyapplicable to the present invention.

The inventors contemplate the fluid output from the cytometer beingmixed with supplementary agents prior to forming the droplets into acryogenic liquid. Some supplementary agents may interfere with theanalysis and sorting of the bioparticles if they are added into the flowcytometer sheath fluid. For example, activated charcoal and dyes such asmalachite green may interfere with cytometric analysis. By introducingsupplementary agents at this stage of the procedure after counting, thisproblem may be overcome.

Freezing the Defined Volume of Liquid to Form a Frozen Ball Containingthe Quantum of the Bioparticle

In this step of the process, the droplets formed in the second stepabove are dropped into a vessel that contains a cryogenic liquid,preferably liquid nitrogen, resulting in the formation of frozen ballsthat contain a desired quantum of selected bioparticle.

This freezing step is preferably performed at a temperature that isoptimal for the preservation of the viability or stability of thebioparticles. It will be appreciated that the optimal temperature mayvary depending on the nature of the bioparticles being prepared.However, those of general skill in the art will readily be able todetermine the optimum temperature range by performing experiments at arange of different temperatures and comparing the numbers ofbioparticles that survive the freezing process. Accordingly, while theuse of liquid nitrogen is preferred, it will be appreciated that anumber of alternative cryogenic liquids may be employed in the inventionin order to satisfy a particular temperature requirement. For example,liquid helium and liquid oxygen are suitable options. Persons skilled inthe field of technology to which the invention relates will readily beable to identify the most appropriate cryogenic liquid to be used basedon the temperature requirements of a particular bioparticle. Inaddition, the temperature of the cryogenic fluid could be controlled byadjusting the pressure of the cryogenic fluid in its holding vessel.

Drying the Frozen Ball to Form Forming a Substantially Dry Solid Product

In accordance with the present invention, the frozen balls formed in thethird step above are then freeze-dried. Freeze-drying may be conductedaccording to standard procedures; (Oetjen, Georg-Wilhelm. Freeze-drying.Wiley-VCH, Weinheim (1999); Rowe, Terence W. G. Edwards freeze-dryinghandbook. Edwards High Vacuum, Crawley (1978); Mellor, J. D.Fundamentals of freeze-drying. Academic Press, London (1978).

As it will be appreciated, it is desirable that this step be conductedin a manner which maintains the viability or stability of thebioparticles. Of course, such conditions may vary depending on thenature of the bioparticles being prepared. However, a person of generalskill in the art will readily be able to determine the most effectiveconditions based on the teachings of Oetjen (1999), Rowe, Terence W. G.Edwards freeze-drying handbook. Edwards High Vacuum, Crawley (1978), andMellor, J. D. Fundamentals of freeze-drying. Academic Press, London(1978).

Optional Quality Control

The inventors contemplate the optional use of a quality control (QC)step following the formation of the product as above described. Such QCmay allow for the minimisation of error in the number of bioparticlespresent within the balls and/or the size of the products produced. QCmay involve either counting the number of bioparticles contained withinselected samples of the product by analytical methods such as culturingthe bioparticles on agar plates, or by flow cytometry, or by nucleicacid-based analytical methods such as the polymerase chain reaction(PCR) within selected samples of the product, or measurement of theuniformity of the product, for example by weighing or measuring the sizeof selected balls.

Following formation of the product in accordance with the invention, theballs may be packaged and stored in a test tube, vial or similarcontainer. Alternatively, the balls can be packaged in blister packssimilar to those used for tablets. The balls are preferably stored underconditions that minimise the exposure of the balls to oxygen andhumidity. Suitable storage conditions include, for example, storageunder vacuum or storage under inert gasses such as nitrogen or argon.Alternatively, a dehumidifying agent such as silica gel can be packagedwith the balls. The balls can be packaged individually or in groups. Forexample, a ball that contains 10 E. coli cells can be packaged in groupsof ten so that each package contains 100 E. coli cells.

Subsequently, the balls may be put to use in a particular application byopening the package that contains the ball and then tipping thecontainer upside down so that is preferably mixed or agitated to assistwith rehydration of the ball. Where the ball is added to a dry sample,such as the surface of an agar plate, a volume of liquid such as watermay then be added to the plate to rehydrate the ball. In the case of theball being contained in a vessel such as a vial or test tube, a sampleof liquid may be added to the vessel and the experimentation or analysisto be conducted carried out in such vessel. While not as preferable, theinvention also contemplates the addition of a liquid to the packagingfollowed by transfer of the sample, containing the rehydrated ball, toan appropriate vessel.

The freeze-dried product prepared according to the invention may besuitable for use in a number of commercial and/or research applications(for example, applications pertaining to microbiology, molecularbiology, cell biology, biochemistry, biotechnology, medicine, and thefood and beverage industry). In particular, in those applications whereit is desirable to have control over the number of bioparticles present.

One particular application to which the balls of the invention may beput is in the testing of water samples for the presence of E. coliTraditionally, during such testing, QC steps are conducted which involveadding a known number of E. coli to a water sample and then analysingthe sample. Current protocols for preparing a QC sample involvepreparing a suspension of E coli and then performing an analysis toestimate the number of E. coli per ml of liquid. An aliquot of thissuspension is then used in the testing procedure. As it will beappreciated, this known procedure may suffer from experimental artefactsdue to the fact that one cannot determine with accuracy the exact numberof E. coli cells present in the sample used, and also based on the lossof E. coli cells due to adhesion to surfaces of manipulation apparatus,such as pipettes. Alternative techniques involve a water sample beingseeded with a freeze-dried sample of E. coli. These freeze-dried E. colisamples, which are commercially available, do not contain accuratenumbers of E. coli and are not in a format that can be easilymanipulated without loss of E. coli cells due to adhesion of cells tosurfaces. The use of the balls of the present invention may introduceaccuracy into QC procedures as precise numbers of organisms will be ableto be added to a quality control sample and not lost duringmanipulation.

A further example of an application to which the balls of the inventionmay be put is in internal quality control (IQC) techniques used inmicrobiology. Such techniques are relatively new. One such procedure isdescribed in PCT/AU00/00896 and involves the addition of an exact numberof modified microorganisms to a sample before analysis. Themicroorganisms are modified to ensure that they can be easilydifferentiated from microorganisms that are present in the sample. Forexample, a green fluorescent protein (GFP) gene may be inserted into theorganisms so that they can be differentiated from microorganisms presentin the sample by their fluorescent properties. Use of the balls of thepresent invention may overcome problems associated with the format inwhich the modified microorganisms are presently used. For example,liquid samples, which may contain accurate numbers of a particularmodified organism, invariably suffer from loss of activity duringmanipulation.

The freeze-dried balls of the present invention may also have specificapplication in delivery systems for oral vaccines. The use of the ballsin this application would enable exact numbers of microorganisms orantigens to be administered to a subject, with no loss of microorganismsor antigens during administration of the vaccine. Similarly, the productof the invention may find application in the delivery of probiotics orprebiotics to a subject.

Further, the inventors contemplate the product of the invention to havedirect application in the in vitro fertilization (IVF) industry. Theproduct of the invention would enable simple manipulation of precisenumbers of sperm, embryos and eggs.

The food and beverage industry is also likely to benefit from the use ofthe balls of the invention, particularly where microorganisms are usedas starter cultures. For example, the fermentation of food, beer andwine would benefit from the use of technology that allows addition ofexact numbers of microorganisms, allowing the introduction of a greaterlevel of reproducibility in the fermentation process.

Biotechnology processes that involve the growth of cells, bacteria orother bioparticles would benefit from the use of the present technology.For example, the production of recombinant proteins from prokaryotic oreukaryotic cells may often be problematic resulting in differing yieldsof protein from one culture to another. While problems may stem from anumber of factors involved in the culturing, expression, and harvestingprocess, the fact that a culture is seeded with an inconsistent quantityof recombinant cells may be considered one such problem. Accordingly,seeding an initial culture with a ball of the present invention, whichball contains a quantum of the recombinant cells, may alleviate onevariable in the process.

The present invention is also suitable for preparing products containinga quantum of one or more genes or parts of genes for molecularbiological applications. The bioparticle may be in the form of a hostmicroorganism, cell vector containing a known number or type of gene ornucleic acid molecule. In this form, viability of the microorganism isnot necessarily required. In some situations, the inventors contemplatethe use of inactivated or dead microorganisms or cells in the solidproduct according to the invention. The product can be used to add adefined number of bioparticle to a gene related assay.

EXAMPLES Example 1 Production of Products Using a Electrostatic CellSorter

Preparation of E. Coli

A strain of E. coli (NCTC 9001) was grown at 37° C. for 24 hours in 1.6%(w/v) tryptone and 1% (w/v) yeast extract at pH 7.2. The cells werediluted 1 in 1000 in filtered (0.22 μm) phosphate buffered saline (PBS)(Sigma Chemical Company, Sydney, NSW) and analysed immediately.

Analysis of E. Coli

The sample of E. coli was analysed using a Becton Dickinson FACStarplusflow cytometer fitted with a 488 nm water cooled argon ion laser. A 100μm nozzle was fitted and the cytometer set up for sorting according tothe manufacturers instructions. Sheath fluid consisted of filtered (0.22μm) PBS plus 4% (w/v) bovine serum albumin fraction V (Sigma ChemicalCompany at pH 7.4.

A region was defined on a scatter plot of Side scatter verses Forwardscatter that contained the E. coli. This region was then used to sortthe E. coli.

Selection of Desired E. Coli

The cytometer was set, according to the manufacturers instructions, tosort samples of 300 E. coli cells.

Freezing and Freeze-Drying the Droplets

Droplets from the cytometer were collected into test tubes thatcontained liquid nitrogen. After collection of the droplets, the tubeswere placed in a Dynavac FD12 freeze dryer and dried overnight at avacuum of 2×10−1 Torr and a condenser temperature of −50° C.

The next day, the freeze-dried particles were removed from the freezedrier and individually placed onto nutrient agar plates (Oxoid,Australia), and 200 μl of sterile 0.9% saline was carefully pipettedonto each ball. The balls were allowed to rehydrate for 5 min and thenspread with a sterile plastic spreader. After incubation at 37° C. for12 hours the agar plates were examined and were observed to containbetween 250 and 300 E. coli colonies.

Example 2 Production of Products that Contain a Quantum of Viable E.Coli Cells

Preparation of E. Coli

A strain of E. coli (NCTC 9001) was grown at 37° C. for 24 hours in 1.6%(w/v) tryptone and 1% (w/v) yeast extract at pH 7.2. The cells werediluted 1 in 1000 in filtered (0.22 μm) PBS and analysed immediately.

Analysis of E. Coli

The sample of E. coli was analysed using a Becton Dickinson FACScaliburflow cytometer that had been modified to enable 30 cells to be dispensedwithin a single droplet. Serum was injected into the droplet whilst itwas forming to enable production of a droplet that freeze dries into aspherical mass without modifying the flow cytometer's sheath fluid.

The cytometer modification involved inserting, into the capture tubewithin the flow cell, a length of hypodermic tubing (A-M Systems,Calsborg, USA). A length of silicon tubing was connected to thehypodermic needle (A-M System, Calsborg, USA). A length of hypodermictubing was connected to other end of the silicon tubing (A-M System,Calsborg, USA). The drop is formed in a droplet nozzle into which thehypodermic tubing from the cytometer is inserted. When the cytometer isturned on, drops are formed at the end of the hypodermic tubing from thenozzle.

A peristaltic pump (Cole Parmer Instruments, Illinios, USA) was used toinject horse serum through length of hypodermic tubing (A-M System,Calsborg, USA), which was also inserted into the droplet nozzle so thatthe serum can be mixed with the output of the flow cytometer to formdrops. The flow rate of the pump was adjusted to match the flow rate ofthe fluid exiting the cytometer sort line.

To control the beginning of droplet formation a micro bore double actingpneumatic cylinder (Asco, Frenches Forest, NSW, Australia) was used withan attached polypropylene vacuum nozzle. The vacuum nozzle attached tothe end of the shaft of the cylinder was held near the droplet nozzleexit point. The vacuum nozzle ensures that all liquids received from thedroplet nozzle are sent to waste before droplet formation begins. Onactivation of the double acting cylinder, the vacuum nozzle is removedtherefore allowing a droplet form.

A region was defined on a scatter plot of Side scatter verses Forwardscatter that contained the E. coli. This region was then used to sortthe E. coli.

Selection of Desired E. Coli

The cytometer was set, according to the manufacturers instructions, tosort samples of 30 E. coli cells. The flow rate and the concentration ofthe E. coli were adjusted to ensure that the sort rate was between 150and 200 sorts per second.

Freezing and Freeze-Drying the Droplets

Droplets from the cytometer were collected into test tubes thatcontained liquid nitrogen. After collection of the droplets, the tubeswere placed in a Dynavac FD12 freeze dryer and dried overnight at avacuum of 2×1 0-1 Torr and a condenser temperature of −70° C.

The next day, the vials were capped under vacuum and removed from thefreeze drier. The vials were opened and the balls individually placedonto nutrient agar plates (Oxoid, Australia), and 200 μl of sterile 0.9%saline was carefully pipetted onto each ball. The balls were allowed torehydrate for 5 min and then spread with a sterile plastic spreader.After incubation at 37° C. for 12 hours the agar plates were examinedand were observed to contain between 25 and 30 E. coli colonies.

Example 3 Production of Balls that Contained a Quantum of BacillusSubtilis Cells Containing a Single Copy of a Green Fluorescent Protein(GFP) Gene

The ability to provide a sample which contains a defined number of geneswill be particularly useful in molecular biological assays. In thissituation, the bioparticle can be a microorganism which harbours a knownnumber of copies of a particular gene. The gene maybe in one or morevectors in the microorganism, or incorporated in the genome of themicroorganism.

Preparation of Bacillus Subtilis

A strain of Bacillus subtilis (1049 Pxyl rpsB-GFP) was grown in nutrientbroth plus 1% (w/v) xylose at 37° C. for 12 hours. This strain containeda single copy of the green fluorescent protein gene (GFP) under thecontrol of a xylose promoter. The sample was diluted 1 in 1000 intofiltered (0.22 μm) PBS plus 1% (w/v) xylose.

Analysis of Bacillus Subtilis

The sample of Bacillus subtilis was analysed using a modified BectonDickinson FACScalibur flow cytometer as in example 2.

A region was defined on a scatter plot of green fluorescence (FL1)verses Forward scatter that contained the Bacillus subtilis. This regionwas then used to sort Bacillus subtilis spores.

Selection of Desired Bacillus Subtilis

The cytometer was set, according to the manufacturers instructions, tosort samples of 30 Bacillus subtilis cells. The flow rate and theconcentration of Bacillus subtilis were adjusted to ensure that the sortrate was between 200 and 220 sorts per second.

Freezing and Freeze-Drying the Droplets

Droplets were collected into glass freeze drying vials that containedliquid nitrogen. After collection of the droplets, the vials were cappedwith the caps halfway inserted and placed in a Dynavac FD12 freeze dryerand dried overnight at a vacuum of 2×10−1 Torr and a condensertemperature of −70° C.

The next day, the freeze-dried balls were removed from the freeze drierand individually placed onto nutrient agar plates (Oxoid, Australia)which had been spiked with 200 μl of a sterile solution of 1% (w/v)xylose. Sterile saline solution (200 μl) was carefully pipetted ontoeach ball. The balls were allowed to rehydrate for 30 seconds and thenspread with a sterile plastic spreader. After incubation at 37° C. for12 hours the agar plates were examined and were observed to containbetween 25 and 30 Bacillus subtilis colonies.

The plates were examined under a UV light and each colony was observedto fluoresce green. This demonstrated that each Bacillus subtilis cellwithin the ball contained the GFP gene.

Example 4 Production of Balls that Contain a Quantum of Viable BacillusCereus Spores

Preparation of B. Cereus Spores

A strain of B. cereus (ATCC 10876) was grown for 7 hours in nutrientbroth (Oxoid, CMI) at 37° C. A 1 ml aliquot of the B. cereus culture wasspread on a nutrient agar plate (Oxoid, Australia) and allowed to dry atroom temperature for 24 hours. Once a visible lawn of growth wasobserved covering the plate, the plate was incubated at 37° C. for 48hours.

The resulting spore culture lawn was removed from the agar plate with a10 μl sterile loop and suspended in 1.5 ml sterile de-ionised water. Thespore suspension was washed four times by repetitive centrifugation(6,200 rpm, 2 minutes) and re-suspended in 1.5 ml de-ionised water. Thefinal spore pellet was suspended in 1 ml Isoton II (Beckman Coulter) andstored at 2-8° C.

A series of dilutions of the spore preparation in Isoton II (Beckmancoulter) were analysed on the Becton Dickinson FACScalibur flowcytometer. The optimal spore concentration was determined by ensuringthe cytometer was detecting between 1500 and 2000 events per second.

Staining of B. Cereus Spores

A 20 μl aliquot of the prepared spore suspension was diluted in 400 μlIsoton II (Beckman Coulter). Syto 11 green fluorescent nucleic acidstain (Molecular Probes, Eugene, USA) was added to the diluted sporesuspension to give a final concentration of 0.0005 mM. The suspensionwas then incubated at room temperature for 30 minutes.

Analysis of B. Cereus

The sample of B. cereus spores was analysed using a modified BectonDickinson FACScalibur flow cytometer as described in example 2.

Selection of Desired B. Cereus

The cytometer was set, according to the manufacturers instructions, tosort samples of 33 B. cereus cells. The flow rate and sort region wereadjusted to ensure that the sort rate was between 200 and 250 sorts persecond.

Freezing and Freeze-Drying the Droplets

Droplets from the cytometer were collected into freeze dry vials thatcontained liquid nitrogen. Control plates were prepared by collectingsingle drops onto nutrient agar plates. This enabled enumeration of theviable spores that were being sorted (see Table 1). The vials containingthe frozen drops were placed in a Dynavac FD12 freeze dryer and driedovernight at a vacuum of 2×10−1 Torr and a condenser temperature of −70°C. The next day the vials were sealed under vacuum and then crimped.

Rehydration and Evaluation

The vials were opened and the freeze-dried balls were individuallytipped onto nutrient agar plates (Oxoid, Australia), and 200 μl ofsterile 0.9% saline was carefully pipetted onto each ball. The ballswere allowed to rehydrate for 1 min and then the plate was rotated toallow the rehydration liquid and dissolved freeze-dried ball to spreadacross the plate. After incubation at 37° C. for 8 hours, the agarplates were examined and were observed to contain between 28 and 33 B.cereus colonies. The mean colony forming unit (cfu) for 15 re-hydratedB. cereus freeze-dried balls was 30.1 with a standard deviation of 1.7(Table 1).

The counts for the control plates were similar to the counts for there-hydrated freeze dried balls (Table 1) indicating that all spores thatwere sorted survived the freeze drying process. The reason that 33viable spores were not sorted every time was probably due to thecytometer occasionally sorting debris material, non-viable spores ornon-viable cells. This could probably be overcome by producing a morepure spore preparation. TABLE 1 Control data and post freeze dryingrecovery of B. cereus from freeze-dried balls Control plates Re-hydratedballs Sample No (cfu) (cfu)  1 30 31  2 31 31  3 31 33  4 30 32  5 30 32 6 28 30  7 31 30  8 29 29  9 30 27 10 31 30 11 27 28 12 27 31 13 31 2814 30 29 15 31 30 Mean 29.8 30.1 Standard 1.4 1.7 deviationStability of B. Cereus Freeze-Dried Balls

The stability of the freeze dried balls that contained B. cereus sporeswas tested by storing the sealed vials at 4° C., 22° C. and 37° C.respectively and then rehydrating the balls on nutrient agar plates. Twodifferent batches were produced and tested (Tables 2 & 3). No reductionin recovery of viable B. cereus was observed after storage for 33 daysat 37° C., after 21 days at 4° C. and after 33 days at 20° C. TABLE 2Stability data for storage at 20° C. and 37° C. Re-hydrated Pre-freezeRe-hydrated Day 33 Sample drying Re-hydrated Day 33 (37° C.) No controls(cfu) Day 0 (cfu) (22° C.) (cfu) (cfu)  1 26 28 30 30  2 25 30 28 25  327 30 30 26  4 29 29 29 29  5 26 30  6 25 28  7 26 29  8 27 30  9 28 2910 22 30 Mean 26.1 29.3 29.3 27.5 Standard 1.9 0.8 0.96 2.4 Deviation %112% 112% 105% survival

TABLE 3 Stability data for storage at 4° C. Pre-freeze dryingRe-hydrated Day 21 Sample No controls (cfu) (cfu)  1 25 27  2 27 24  329 27  4 24 26  5 29 26 Mean 26.8 26 Standard 2.3 1 deviation % survival97%

Example 5 Production of Balls that Contained a Quantum of 8 μm BeadsCoated with Viable E. Coli

Preparation of E. Coli

A strain of E. coli (NCTC 9001) was grown at 37° C. for 24 hours in 1.6%(w/v) tryptone and 1% (w/v) yeast extract pH 7.2. The cells were washedin filtered (0.22 μm) PBS and processed immediately.

Preparation of Beads

Amino modified magnetic beads with a diameter of 8 microns were suppliedby Spherotch Inc. (Libertyville, Ill., USA). A 1 ml aliquot of the beadswas washed three times in PBS with a magnetic concentrator (Spherotech)according to the manufacturers instructions. The beads were resuspendedin 35% (v/v) glutaraldehyde (Sigma) in PBS, vortexed for 2 minutes andincubated at room temperature on a rotary mixer for 2 hours. The beadswere then washed four times in PBS, resuspended in 1 ml of PBS andvortexed for 2 minutes.

Coating Beads with E. Coli

A 20 μl aliquot of the bead preparation was mixed with 1 ml of washed E.coli cells at an approximate concentration of 1×10⁸ per ml. The samplewas then incubated on a rotary mixer at room temp for 30 minutes.

Staining E. Coli Coated Beads

A 20 μl aliquot of the prepared spore suspension was diluted in 400 μlIsoton II (Beckman Coulter). Syto 16 green fluorescent nucleic acidstain (Molecular Probes, Eugene, USA) was added to the diluted sporesuspension to give a final concentration of 0.0005 mM. The suspensionwas then incubated at room temperature for 30 minutes.

Analysis of Coated Beads

The stained bead sample was analysed using a modified Becton DickinsonFACScalibur flow cytometer as in Example 2.

A region was defined on a scatter plot of green fluorescence (FL1)verses Forward scatter that contained the E. coli coated beads.

Selection of Desired Bacillus Subtilis

The cytometer was set, according to the manufacturers instructions, tosort samples of 30 E. coli coated beads. The flow rate and theconcentration of the stained beads were adjusted to ensure that the sortrate was between 200 and 220 sorts per second.

Freezing and Freeze-Drying the Droplets

Droplets were collected into glass freeze drying vials that containedliquid nitrogen. After collection of the droplets, the vials were cappedwith the caps halfway inserted and placed in a Dynavac FD12 freeze dryerand dried overnight at a vacuum of 2×10−1 Torr and a condensertemperature of −70° C.

The next day, the freeze-dried balls were removed from the freeze drierand rehydrated, incubated and the colonies counted as described inExample 2.

After incubation at 37° C. for 12 hours the agar plates were examinedand were observed to contain between 25 and 30 E. coli colonies.

Example 6 Production of Balls that Contained a Quantum of BacillusCereus Spores Produced Without Using a Flow Cytometer

Preparation of B. Cereus Spores

A strain of B. cereus (ATCC 10876) was grown on nutrient agar (Oxoid,Australia) at 37° C. for 24 hours and then kept at room temperature for1 week. Spores were washed from the culture plate with PBS and stored at4° C.

Dilution of Spores

Spores were diluted in PBS to an approximate concentration of 1×10⁷spores per ml and stored at 4° C. This stock dilution was then seriallydiluted in horse serum and a burette was used to place 1 drop from eachdilution onto three nutrient agar plates. The burette was used toproduce drops because drops with a more reproducible volume were foundto be obtained than when pastuer pipettes were used.

The drop was spread using a plastic spreader, the plates incubated at37° C. overnight and colonies counted. The colony counts were used tocalculate the dilution of the spore stock that was required to produceapproximately 30 colonies in one drop.

Production of Balls

The spore stock was diluted in horse serum to a concentration that wasexpected to produce 30 cfu in a single drop. Drops of the diluted sporesuspension were dropped into a bowl of liquid nitrogen and allowed tofreeze. Frozen balls were transferred to freeze drying vials and freezedried as detailed in Example 3.

Rehydration and Evaluation

The next day, the freeze-dried balls were removed from the freeze drierand individually placed onto nutrient agar plates (Oxoid, Australia),and 200 μl of sterile 0.9% saline was carefully pipetted onto each ball.The balls were allowed to rehydrate for 1 min and then the plate wasrotated to allow the rehydration liquid and dissolved freeze-dried ballto spread across the plate. After incubation at 37° C. for 8 hours, theagar plates were examined and the number of colonies recorded (Table 4).The plates were observed to contain between 30 and 58 colonies. The meancfu for 15 re-hydrated B. cereus freeze-dried balls was 40.3 with astandard deviation of 7.8. TABLE 4 Colonies 30 33 42 40 36 44 44 39 3958 44 54 41 31 30 Mean 40.3 Std Dev 7.8Stability of B. Cereus Freeze-Dried Balls

Freeze-dried balls containing a mean of 30 B. cereus spores producedbetween 93 and 99% recovery of spores. Two batches of B. cereusfreeze-dried balls produced 93% spore recovery after 33 days at 37° C.,97% spore recovery after 21 days at 4° C. and 99% spore recovery after33 days at 20° C.

Example 7 Production of a Large Quantum of E. Coli

Preparation of E. Coli

A strain of E. coli (ATCC 11775) was grown in nutrient broth (Oxoid,Australia) at 37° C. for 12 hours.

Freezing and Freeze-Drying the Droplets

A glass pasteur pipette was used to drop droplets of E. coli brothculture into a beaker of liquid nitrogen. The frozen droplets werecollected using a sieve and placed into a chilled (−20° C.) glassfreeze-drying vessel.

The frozen droplets were placed in a Dynavac FD12 freeze dryer and driedovernight at a vacuum of 2×10−1 Torr and a condenser temperature of −70°C.

The next day, the freeze-dried balls were removed from the freeze drierand individually placed into nutrient broth (as detailed above). Afterincubation at 37° C. for 12 hours the tubes were examined and wereobserved to contain viable cultures of E. coli.

Comments

The technology of the present invention may be put to applications whichmay not require a small quantum of the bioparticles to be present ineach ball or within a group of balls. One such example is in oralvaccination applications. In such application balls containing anapproximate number of bioparticles (e.g. between 5×10⁵ and 1×10⁶) may beused.

According to the present embodiment, frozen balls containing a quantumof bioparticles are formed by allowing droplets of the suspension ofbioparticles to fall into a cryogenic liquid as described above. Thedroplets may be formed by forcing an appropriate suspension ofbioparticles through any suitable orifice. For example, this step may beperformed using a peristaltic pump connected to a pastuer pipette. Thoseskilled in the art will appreciate alternative means by which suchdroplets may be formed. Where a flow cytometry apparatus has beenemployed to analyse and select the bioparticles, quantities ofbioparticles may be expressed therefrom by known means.

As with the previously described embodiment of the invention, thedroplets which are formed are allowed to fall into a cryogenic liquid toform a frozen ball of bioparticles.

The frozen balls contained within the cryogenic liquid may then besubjected to a freeze drying procedure as described above to form theballs of the invention. Freeze-dried balls may be packaged, stored, andprepared for use as previously described.

As will be appreciated, suspension and growth media's, temperatures, andother conditions of the process according to the present embodiment ofthe invention may be optimised to the needs of a particular bioparticleand the application to which the final product may be put. Generally,the conditions should be such that the viability or stability of thebioparticles is maintained during processing.

As with the previously described embodiment of the invention, qualitycontrol steps may be conducted post formation of the balls produced inaccordance with the present embodiment. In this form of the invention,quality control steps may include: counting the number of bioparticlescontained within selected samples of the product by analytical methodssuch as culturing the bioparticles on agar plates, or by flow cytometry,or by nucleic acid based analytical methods such as the polymerase chainreaction (PCR), or measurement of the uniformity of the product, forexample by weighing or measuring the size of selected balls.

The product of this alternative embodiment of the invention may be usedin various applications, for example vaccination applications,microbiological applications, or applications pertaining to molecularbiology, where an estimate of the number of bioparticles within theproduct is sufficient to reach a desired end.

The invention has been described herein, with reference to certainpreferred embodiments, in order to enable the reader to practice theinvention without undue experimentation. However, a person havingordinary skill in the art will readily recognise that many of thecomponents and parameters may be varied or modified to a certain extentwithout departing from the scope of the invention. Furthermore, titles,headings, or the like are provided to enhance the reader's comprehensionof this document, and should not be read as limiting the scope of thepresent invention.

The present invention allows the production of batches of products thatcontain a known and substantially identical number of a bioparticle. Thepresent inventors have consistently shown the method of the inventioncan produce a product having a known number of a bioparticle withinaround 7% standard deviation. In addition, the product in itsfreeze-dried form can be handled easily without the concern of loss orshedding of the bioparticle present. To use or retrieve the bioparticlefrom the product, all that is required is to add a liquid to theproduct. In order to ensure that the actual number of bioparticle isused, the product can be added in its dry form to a test and thenrehydrated with a suitable liquid. The product can be moved to differentvessels in its dry form without the concern that some of the bioparticlewill remain in the original vessel. This is a clear advantage over theprior art.

It will be appreciated by persons skilled in the art that numerousvariations and/or modifications may be made to the invention as shown inthe specific embodiments without departing from the spirit or scope ofthe invention as broadly described. The present embodiments are,therefore, to be considered in all respects as illustrative and notrestrictive.

1-52. Canceled
 53. A process for forming a product containing a definedsmall number of microorganisms, the process comprising: providingmicroorganisms selected from the group consisting of viruses, bacteria,yeast, fungi, parasites, protozoa, cells and mixtures thereof insuspension; selecting a defined number of the microorganisms of between1 and 1000 from the suspension by means to sense a microorganism;capturing the defined number of microorganisms in a frozen body; anddrying the frozen body to produce a product containing the definednumber of microorganisms, wherein the product is capable of beingtransferred between containers in its solid form without substantialloss of the microorganisms, wherein the product is capable of releasingthe microorganisms in a liquid, and wherein the defined number of themicroorganisms in the product when measured in two or more replicates iswithin a standard deviation of 10% or less of the defined number. 54.The process according to claim 53 wherein the sensing means is selectedfrom the group consisting of flow cytometer, absorption at a particularwavelength, density, magnetism, specific gravity, impedance, ability toscatter light, luminescence, fluorescence, Coulter sensing, and ramanmicroscopy.
 55. The process according to claim 54 wherein the sensingmeans is a flow cytometer.
 56. The process according to claim 53 whereinthe bacteria are selected from the group consisting of Legionella,Salmonella, Leptospirosis, Escherichia, Saccharomyces, Clostridium,Vibrio, Pseudomonas, Bacillus, Streptomyces, Staphylococcus,Enterobacter, Listeria, Candida, Zygosaccharomyces, and mixturesthereof.
 57. The process according to claim 56 wherein the bacteria areEscherichia coli or Bacillus cereus.
 58. The process according to claim53 wherein the parasites are selected from the group consisting ofCryptosporidium, Giardia, Cyclospora, Toxoplasma, Eimeria, and mixturesthereof.
 59. The process according to claim 53 wherein themicroorganisms are viable in the product.
 60. The process according toclaim 53 wherein the suspension of microorganisms is selected from thegroup consisting of a microbial culture, a suspension of cells, asuspension of particles including one or more microorganisms, andmixtures thereof.
 61. The process according claim 60 wherein themicroorganism contains a desired amount of a protein, carbohydrate,polysaccharide, gene or a nucleic acid molecule.
 62. The processaccording to claim 61 wherein microbial culture is a bacterial culture.63. The process according to claim 53 wherein the microorganisms arecaptured in a volume from 0.001 ml to 1 ml prior to freezing.
 64. Theprocess according to claim 63 wherein the volume is about 0.1 ml. 65.The process according to claim 53 wherein the defined number of themicroorganisms in the product is within a standard deviation of 5% orless.
 66. The process according to claim 65 wherein the defined numberof the microorganisms in the product is within a standard deviation of2%.
 67. The process according to claim 53 wherein the product is formedby snap-freezing a volume of liquid containing the defined number ofmicroorganisms and then drying the frozen body to form the product. 68.The process according to claim 67 wherein the snap-freezing is carriedout by placing the volume of liquid containing the defined number ofmicroorganisms into a cryogenic liquid.
 69. The process according toclaim 68 wherein the cryogenic liquid is selected from the groupconsisting of liquid nitrogen, liquid helium, liquid oxygen, andmixtures thereof.
 70. The process according to claim 69 wherein thecryogenic liquid is liquid nitrogen.
 71. The process according to claim68 wherein the cryogenic liquid is placed in a container, a dropletcontaining the defined number of microorganisms is placed in thecontainer to form the frozen body, and the container holding the frozenbody is then subjected to freeze-drying to form a substantially drysolid product in the container.
 72. The process according to claim 71wherein after drying, the container is capped or sealed for storage andtransport of the product.
 73. The process according to claim 53 whereinthe product is a small roundish mass in the form of a ball or sphere.74. The process according to claim 53 wherein the product contains adefined number of microorganisms of between 10 and
 100. 75. The processaccording to claim 74 wherein the product contains a defined number ofmicroorganisms of
 30. 76. The process according to claim 53 furthercomprising selecting a desired type of microoganism from a mixture ofmicrooganism types.
 77. The process according to claim 53 wherein oneproduct forms a desired number of microorganisms.
 78. The processaccording to claim 66 wherein two or more products form the desirednumber of microorganisms.
 79. The process according to claim 53 furthercomprising providing one or more supplementary agents.
 80. The processaccording to claim 79 wherein the supplementary agent is selected fromthe group consisting of sucrose, trehalose, lactose, maltose, glucose,galactose, raffinose, fructose, xylose, cellobiose, gelatin, xanthamgum, guar gum, maltodextrans, polyethylene glycol, dextran, polyvinylpyrrolidone, sodium thiosulfate, activated charcoal, ascorbic acid,ascorbate peroxidase, glutathione reductase, peroxiredoxin, sodiumglutamate, proline, potassium glutamate, proline betaine, glycinebetaine, skim milk, serum, trypsin, peptone, tryptone, yeast extract,soy protein, meat extract, mannitol, glycerol, sorbitol, inositol,butanol, tertiary butyl alcohol, honey, sodium acetate, myo-inositol,calcium chloride, whey, hydroxyectoine, ectoine, and mixtures thereof.81. The process according to claim 53 further comprising a qualitycontrol step to determine the number of microorganisms contained in oneor more products, and/or to measure uniformity of the product.
 82. Theprocess according to claim 81 wherein the quality control step isgrowing microoganisms from the product and confirming the actual numberof microorganisms in the product.
 83. A process for forming a solidproduct containing a defined small number of a viable bacteria, theprocess comprising: providing bacteria in suspension; selecting adefined number of the bacteria of between 5 and 100 in a drop using aflow cytometer to sense a bacterium; adding the drop containing thebacteria to liquid nitrogen to form a frozen ball containing the definednumber of the bacteria; and subjecting the frozen ball to freeze-dryingto form a substantially dry solid product containing the defined numberof the bacteria, wherein the product is capable of being transferredbetween containers in its solid form without loss of the bacteria, andwherein the product is capable of releasing the defined number of viablebacteria in a liquid, and wherein the defined number of the bacteria inthe product when measured in two or more replicates is within a standarddeviation of less than about 5% of the desired number of bacteria. 84.The process according to claim 83 wherein the bacteria are selected fromthe group consisting of Legionella, Salmonella, Leptospirosis,Escherichia, Saccharomyces, Clostridium, Vibrio, Pseudomonas, Bacillus,Streptomyces, Staphylococcus, Enterobacter, Listeria, Candida,Zygosaccharomyces, and mixtures thereof.
 85. The process according toclaim 83 wherein the bacteria are Escherichia coli or Bacillus cereus.86. The process according to claim 83 wherein the microorganism containsa desired amount of a protein, carbohydrate, polysaccharide, gene or anucleic acid molecule.